David Bikard

Institut Pasteur · Department of Microbiology

Transfer RNA molecules have been recently recognized as widespread targets of bacterial immune systems. Translation inhibition through tRNA cleavage or modification inhibits phage propagation, thereby protecting the bacterial population. To counteract this, some viruses encode their own tRNA molecules, allowing infection to take place. The AriB eff...

Background Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing...

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to transloca...

Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In thi...

Viruses compete with each other for limited cellular resources, and some deliver defence mechanisms that protect the host from competing genetic parasites¹. The phage antirestriction induced system (PARIS) is a defence system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB)². Howeve...

Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health¹. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells², we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle...

Integrons are genetic systems that accelerate bacterial adaptation by acquiring and shuffling gene cassettes. Mobile integrons spread antibiotic resistance genes among bacteria, while the sedentary chromosomal integrons contain up to hundreds of cassettes of unknown function. Here, we show that many of these cassettes encode anti-phage defence syst...

Vibrio cholerae O1 El Tor , the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools exist. While CRISPRi has been applied in V. cholerae , improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium. In this s...

CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and...

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to transloca...

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, po...

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble in...

We present here an approach to protein design that combines (i) scarce functional information such as experimental data (ii) evolutionary information learned from a natural sequence variants and (iii) physics-grounded modeling. Using a Restricted Boltzmann Machine (RBM), we learn a sequence model of a protein family. We use semi-supervision to leve...

Type IV-A CRISPR-Cas systems are primarily encoded on plasmids and form multi-subunit ribonucleoprotein complexes with unknown biological functions. In contrast to other CRISPR-Cas types, they lack the archetypical CRISPR acquisition module and encode a DinG helicase instead of a nuclease component. Type IV-A3 systems are carried by large conjugati...

We present here an approach to protein design that combines evolutionary and physics-grounded modeling. Using a Restricted Boltzmann Machine, we learned a sequence model of a protein family and propose a strategy to explore the protein representation space that can be informed by external models such as an empirical force field method (FoldX). This...

Computational protein design facilitates the discovery of novel proteins with prescribed structure and functionality. Exciting designs were recently reported using novel data-driven methodologies that can be roughly divided into two categories: evolutionary-based and physics-inspired approaches. The former infer characteristic sequence features sha...

Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9’s application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies...

Antimicrobial resistance (AMR) in bacteria is a major threat to public health; one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been studied extensively in vitro but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here,...

Microbiome research is revealing a growing number of bacterial genes that impact our health. While CRISPR-derived tools have shown great success in editing disease-driving genes in human cells, we currently lack the tools to achieve comparable success for bacterial targets. Here we engineer a phage-derived particle to deliver a base editor and modi...

Computational protein design facilitates discovery of novel proteins with prescribed structure and functionality. Exciting designs were recently reported using novel data-driven methodologies that can be roughly divided into two categories: evolutionary-based and physics-inspired approaches. The former infer characteristic sequence features shared...

Integrons are adaptive bacterial devices that rearrange promoter less gene cassettes into variable ordered arrays under stress conditions, to sample combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a...

While studying CRISPR‐Cas function in prokaryotic immunity against foreign DNA, researchers realized that when programmed to target the bacteria's own chromosome, these systems would kill the cell. The problem of antimicrobial resistance has been well documented, beginning only shortly after the implementation of the first chemical antimicrobials....

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage–host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing...

Antibiotic resistance (AMR) in bacteria is a major threat to public health, and one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been extensively studied in vitro , but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Her...

Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E. coli P2-like phages and their parasitic P4-like satellites carry hot...

Prokaryotes have numerous mobile genetic elements (MGEs) that mediate horizontal gene transfer (HGT) between cells. These elements can be costly, even deadly, and cells use numerous defense systems to filter, control, or inactivate them. Recent studies have shown that prophages, conjugative elements, their parasites (phage satellites and mobilizabl...

Prokaryotes have numerous mobile genetic elements (MGE) that mediate horizontal gene transfer between cells. These elements can be costly, even deadly, and cells use numerous defense systems to filter, control or inactivate them. Surprisingly, many phages, conjugative plasmids, and their parasites, phage satellites or mobilizable plasmids, encode d...

Viruses that infect bacteria (phages) are increasingly recognized for their importance in diverse ecosystems but identifying and annotating them in large-scale sequence datasets is still challenging. Although efficient scalable virus identification tools are emerging, defining the exact ends (termini) of phage genomes is still particularly difficul...

Motivation: Modeling of protein family sequence distribution from homologous sequence data recently received considerable attention, in particular for structure and function predictions, as well as for protein design. In particular, Direct Coupling Analysis, a method to infer effective pairwise interactions between residues, was shown to capture i...

A Correction to this paper has been published: https://doi.org/10.1038/s41564-021-00889-w.

A Correction to this paper has been published: https://doi.org/10.1038/s41564-021-00893-0.

Integral outer membrane proteins (OMPs) are crucial for the maintenance of the proteobacterial envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OM proteins (OMPs) activates the sigmaE (σ E ) transcriptional response. σ E upregulates OMP-biogenesis factors, including the b-barrel...

Bacteria from the same species can differ widely in their gene content. In Escherichia coli, the set of genes shared by all strains, known as the core genome, represents about half the number of genes present in any strain. Although recent advances in bacterial genomics have unravelled genes required for fitness in various experimental conditions,...

The vast expansion of protein sequence databases provides an opportunity for new protein design approaches which seek to learn the sequence-function relationship directly from natural sequence variation. Deep generative models trained on protein sequence data have been shown to learn biologically meaningful representations helpful for a variety of...

Motivation: Modeling of protein family sequence distribution from homologous sequence data recently received considerable attention, in particular for structure and function predictions, as well as for protein design. In particular, Direct Coupling Analysis, a method to infer effective pairwise interactions between residues, was shown to capture im...

The arms race between bacteria and phages led to the emergence of a variety of genetic systems used by bacteria to defend against viral infection, some of which were repurposed as powerful biotechnological tools. While numerous defense systems have been identified in genomic regions termed defense islands, it is believed that many more remain to be...

In Gram-negative bacteria, coordinated remodelling of the outer membrane (OM) and the peptidoglycan is crucial for envelope integrity. Envelope stress caused by unfolded OM proteins (OMPs) activates sigmaE (σ E ) in Enterobacteria. σ E upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyzes OMP-folding. El...

Recent advances in genomics have uncovered the tremendous diversity and richness of microbial ecosystems. New functional genomics methods are now needed to probe gene function in high-throughput and provide mechanistic insights. Here, we review how the CRISPR toolbox can be used to inactivate, repress or overexpress genes in a sequence-specific man...

Recombination systems are widely used as bioengineering tools, but their sites have to be highly similar to a consensus sequence or to each other. To develop a recombination system free of these constraints, we turned toward attC sites from the bacterial integron system: single-stranded DNA hairpins specifically recombined by the integrase. Here, w...

Bacteria from the same species can differ widely in their gene content. In E. coli , the set of genes shared by all strains, known as the core genome, represents about half the number of genes present in any strain. While recent advances in bacterial genomics have enabled to unravel genes required for fitness in various experimental conditions at t...

The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity fo...

The design of novel proteins with specified function and controllable biochemical properties is a longstanding goal in bio-engineering with potential applications across medicine and nanotechnology. The vast expansion of protein sequence databases over the last decades provides an opportunity for new approaches which seek to learn the sequence-func...

CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic detai...

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell...

CRISPR-Cas systems provide RNA-guided adaptive immunity to the majority of archaea and many bacteria. They are able to capture pieces of invading genetic elements in the form of novel spacers in an array of repeats. These elements can then be used as a memory to destroy incoming DNA through the action of RNA-guided nucleases. This chapter describes...

Prokaryotes use CRISPR-Cas systems for adaptive immunity, but the reasons for the frequent existence of multiple CRISPRs and cas clusters remain poorly understood. Here, we analysed the joint distribution of CRISPR and cas genes in a large set of fully sequenced bacterial genomes and their mobile genetic elements. Our analysis suggests few negative...

Abstrac Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli , two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell, the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-l...

The catalytic null mutant of the Cas9 endonuclease from the bacterial CRISPR immune system, known as dCas9, can be guided by a small RNA to bind DNA sequences of interest and block gene transcription in a strategy known as CRISPRi. This powerful gene silencing method has already been used in a large number of species and in high throughput screens....

Prokaryotes use CRISPR-Cas for adaptive immunity, but the reasons for the existence of multiple CRISPR and cas clusters remain poorly understood. We found that more than 40% of the genomes encoding a system show atypical genetic organizations. Their analysis revealed negative and positive epistatic interactions between Cas subtypes. The latter ofte...

Our bodies are colonized by a complex ecosystem of bacteria, unicellular eukaryotes and their viruses that together play a major role in our health. Over the past few years tools derived from the prokaryotic immune system known as CRISPR-Cas have empowered researchers to modify and study organisms with unprecedented ease and efficiency. Here we dis...

The absence of CRISPR-Cas systems in more than half of the sequenced bacterial genomes is intriguing, because their role in adaptive immunity and their frequent transfer between species should have made them almost ubiquitous, as is the case in Archaea. Here, we investigate the possibility that the success of CRISPR-Cas acquisition by horizontal ge...

High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs whic...

sgRNAs with off-targets in essential genes induce a strong fitness defect. (A) A sgRNA targeting mdfA has a 10-nt perfect match to rpsL. (B) A sgRNA targeting flgC has a 10-nt perfect match to lptG. (A,B) Matched base pairs are shown in blue. On the plots, sgRNAs targeting the +1 strand or the -1 strand are shown in red or blue respectively. A blac...

Unexpected fitness defects shed light on atypical gene organizations. (A) yejL is located upstream of the sensitive essential gene yejM. (B) ypaB contains the promoter of the essential gene nrdA. (C) sroG is a riboswitch controlling the expression of the essential gene ribB. (D) yceQ is annotated as essential but is actually located upstream rne in...

Predicted internal promoters within operons. We identified 7 operons in which an expected polar effect is not observed, i.e guides targeting a gene upstream of an essential or near-essential gene are not depleted. This suggests that the downstream gene can be expressed from an internal promoter. Promoters predicted from a recent transcription start...

Replacement of genes alsK, bcsB and entD by a Kanamycin resistance cassette through λ-red recombination shows that these genes are not essential. The kanamycin resistance cassette from plasmid pKD4 was amplified with primers designed to introduce 50 bp-long homologies with regions flanking genes alsK, bcsB and entD. Primer couples P1 + P2, P1’ + P2...

Phage λ can package plasmid psgRNAcos upon infection. (A) MG1655 carrying psgRNAcos was infected with λ at MOI = 1. The lysate containing a mix of phage and packaged cosmid was extracted after 2 h and the relative concentrations of phage and cosmid were measured by plaque assay and by transduction into strain MG1655::λ respectively. This strain car...

List of sgRNAs with log2FC values after the growth-based screen in strain LC-E75. Each sgRNA is indexed with its position, orientation (ori), target strand (coding) and computed fold change (log2FC, padj and gamma). Information on the targeted gene is also provided: name (gene), essentiality (essential), position (gene_left, gene_right) and orienta...

List of genes with median log2FC values after growth-based screen. The following information is provided. Gene name (gene), essentiality (essential), orientation (gene_ori), position (gene_left, gene_right), median log2FC for each strand (median_coding and median_template) and median absolute deviation (mad_coding and mad_template), number of guide...

Target regions of guides that produce a strong fitness defect while targeting multiple positions. (A) 348/1932 sgRNAs that simultaneously target several positions induce a fitness defect (log2FC < -2). These guides mostly target rRNAs, tRNAs, repeat regions or elongation factor genes (tufA-tufB). (B) Among sgRNAs targeting REP elements, sgRNAs simu...

Inefficient dCas9-mediated repression of genes regulated by negative feedback loops. lexA and rho are two well-known essential genes whose product inhibits their own expression and which are classified as nonessential in our screen. (A) Relative lexA or rho expression was measured in presence of a lexA- or rho-targeted sgRNA respectively, with or w...

Strains LC-E75 and FR-E01 produce similar concentrations of dCas9. Strains LC-E75 and FR-E01 were grown overnight and diluted 1:100 with aTc for 2 h before harvesting. Samples were run in NuPAGE Novex Bis-Tris gels in reducing condition before transfer to PVDF membranes. Rabbit monoclonal CRISPR-Cas9 antibody and rabbit polyclonal RecA antibody wer...

sgRNAs in the library with a perfect match in λ phage genome. sgRNAs targeting the positive or negative strand are dotted in red or blue respectively. Genes colored in yellow, orange and red respectively correspond to early right, early left and late operon. (PNG)

List of genes with estimates and FDR after transduction screen. Each gene is indexed with its information (gene, essential), operon information (operon) and number of guides targeting the coding strand used for calculations (sgRNAs). For each gene, a linear model was built to take the effect on cell fitness into account (see Methods). The regressio...

List of primers used for qPCR. (DOCX)

Gene interaction network of the top 100 genes having the lowest median log2FoldChange. The STRING database was used to compute a gene interaction network [94]. Genes were colored by function. Line thickness indicates confidence of the interaction. (PNG)